Targeted Isolation and Identification of Sialylated Glycoproteins in Cancer Tissues, Cells and Biofluids

糖蛋白是位于细胞表面和细胞内的大部分蛋白质。 细胞外环境。在癌变过程中，这些糖蛋白的正常功能 细胞间黏附的结合配体、细胞外基质分子、信号受体和 免疫细胞会发生变化。转移进展中的许多步骤涉及重组。 以及细胞外基质中的糖蛋白的降解。因此，糖链和糖蛋白 抗原仍然是FDA批准的癌症生物标志物中的大多数。我们的团队 最近开发了多个N-连接的葡聚糖成像质谱仪工作流程，以 表征临床相关肿瘤组织、生物体液和细胞中的糖类，以及 通过免疫捕获单独靶向糖蛋白。在本提案中，新点击的应用 特异性靶向2，3唾液酸抗原的化学试剂，如唾液酸路易斯A/CA19-9， Sialyl Lewis X和StrA将用于改进肿瘤相关基因的分离和测序 来自FFPE组织、生物液和细胞的糖肽。标准蛋白水解酶与新型ECM靶向研究 分析中将使用蛋白水解酶。为此，本文提出了三个具体目标：1. 结论：1.临床FFPE癌组织中唾液酸糖蛋白的分离、鉴定及共定位； 分离鉴定血浆癌组织中的唾液酸糖蛋白；3.分离鉴定 高水平2，3唾液酸抗原的癌细胞株中的唾液酸糖蛋白。这个 所开发的碳水化合物靶向点击捕捉试剂可直接应用于任何FFPE组织， 通过简单的化学反应获得感兴趣的细胞或生物流体。开发的工具组合 这将有助于鉴定相关的特定糖肽和糖蛋白种类 癌症样本可以应用于癌症预防、进展、 预后和生物标记物的应用。

Glycoproteins account for the majority of the proteins located at the cell surface and in the extracellular environment. During carcinogenesis the normal function of these glycoproteins as binding ligands for cell-cell adhesion, extracellular matrix molecules, signaling receptors, and immune cells becomes altered. Many of the steps in metastatic progression involve re-organization and degradation of the glycoproteins in the extracellular matrix. Hence, glycan and glycoprotein antigens still comprise the majority of FDA approved diagnostic cancer biomarkers. Our group has recently developed multiple N-linked glycan imaging mass spectrometry workflows to characterize the glycomes in clinically relevant tumor tissues, biofluids and cells, as well as individually targeted glycoproteins by immunocapture. In this proposal, application of a new click chemistry reagent specific for targeting of 2,3 sialic acid antigens, like sialyl Lewis A/CA19-9, sialyl Lewis X and sTRA, will be used to improve isolation and sequencing of tumor-associated glycopeptides from FFPE tissues, biofluids and cells. Standard proteases and new ECM-targeted proteases will be used in the analyses. To accomplish this, three Specific Aims are proposed: 1. Isolate, identify and co-localize sialic acid glycoproteins from clinical FFPE cancer tissues; 2. Isolate and identify sialic acid glycoproteins in plasma cancer samples; 3. Isolate and identify sialic acid glycoproteins in cancer cell lines with high levels of 2,3 sialic antigens. The carbohydrate-targeted click capture reagents developed can be applied directly to any FFPE tissue, cell or biofluid of interest using simple chemical reactions. The combination of tools developed herein will facilitate the identification of specific glycopeptide and glycoprotein species in relevant cancer samples which can be applied across the spectrum of cancer prevention, progression, prognosis, and biomarker applications.